Streptomyces are filamentous Gram-positive soil bacteria with a nucleotide base composition greater than 70 mole % G+C (Stackebrandt and Woese, 1981). They produce a wide array of biologically active compounds including over two thirds of the commercially important natural product metabolites (Alderson et al., 1993; Bevax, 1998). Genetic information accumulated over the past 15 years has demonstrated that genes encoding enzymes for natural product assembly are clustered on the Streptomyces genome (Martin, 1992). In addition, one or more pathway-specific transcriptional regulatory genes, and at least one resistance gene are typically found within the antibiotic biosynthetic gene cluster (Chater, 1992). Heterologous hybridization with gene probes based on highly conserved biosynthetic enzyme amino acid sequences has been useful to clone antibiotic biosynthetic genes (Hopwood, 1997; Seno and Baltz, 1989; Turgay and Marahiel, 1994).
The mitomycins are a group of natural products that contain a variety of functional groups, including aminobenzoquinone and aziridine ring systems. One representative of the family, mitomycin C (MC), was the first recognized bioreductive alkylating agent. In particular, since its discovery and demonstration of anticancer activity in the 1960s, many aspects of the chemistry and biology of MC have been investigated. This has provided detailed information on its unprecedented molecular mechanism, unique biological and pharmacological properties, drug resistance, and bioactive analogues (Hata et al., 1956; Verweij, 1997). MC is regarded as the prototype natural product alkylating agent whose activity is dependent on the reductive activation (either chemically, such as low pH, or enzymatically, such as DT-diaphorase, NADH cytochrome c reductase) (Boxer, 1997; Cummings et al., 1998). Activated MC crosslinks double-stranded DNA, which in turn induces diverse biological effects including selective inhibition of DNA synthesis, mutagenesis, induction of DNA repair (SOS response), sister chromatid exchange, signal transduction, and induction of apoptosis (Tomasz and Palem, 1997). Tumor hypoxia and the increased expression of bioreductive enzymes in malignant cells create a selective environment for drug activation and make MC an attractive agent for anti-tumor therapy (Spanswick et al., 1998). MC has become one of the most effective antitumor drugs against non-small cell lung carcinoma and other soft tumors, as well as a clinically important component of combination cancer chemotherapy and radiotherapy of solid tumors (Henderson, 1993).
In addition to its biological and pharmacological importance, MC is prominent because its molecular mechanism represents a model for structurally related antitumor antibiotics such as porfiromycin (Pan and Iracki, 1988), mitiromycin (Wakaki et al., 1958), FR66979 (Paz and Hopkins, 1997), FR900482 (Williams et al., 1997), FK973 (Hirai et al., 1994), and FK317 (Naoe et al., 1998), as well as structurally unrelated bioreductive agents such as EO9 (Smitskampwilms et al., 1996), and tirapazamine (Evans et al., 1998). Numerous MC derivatives have been synthesized and tested for enhanced activities, including the recently identified selective protein tyrosine kinase inhibitor, 1a-docosahexaenoyl MC (Kasai and Arai, 1995; Shikano et al., 1998).
Streptomyces lavendulae produces MC. The molecule has an unusual structure comprised of aziridine, pyrrolizidine, pyrrolo-(1,2a)-indole, and amino-methylbenzoquinone rings to give the mitosane nucleus (Webb et al., 1962). The mitosane core of MC was shown to be derived from the junction of an amino-methylbenzoquinone (mC7N unit) and hexosamine (C6N unit) (Hornemann, 1981). The C6N unit consists of carbons 1, 2, 3, 9, 9a, 10, with the aziridine nitrogen derived intact from D-glucosamine (Hornemann et al., 1974).
The mC7N unit in MC and the ansamycins is derived from 3-amino-5-hydroxybenzoic acid (AHBA) (Becker et al., 1983; Kibby and Richards, 1981). AHBA was first shown to be incorporated into the ansamycin antibiotic actamycin (Kibby et al., 1980). Subsequently, it was confirmed as an efficient precursor for rifamycin (Becker et al., 1983; Kibby and Rickards, 1981; Ghilsalba and Neuesch, 1981), geldanamycin (Potgieter, 1983), ansamitocin (Hatano et al., 1982), ansatrienin (Wu et al., 1987), streptovaricin (Staley and Rinehart, 1991) and naphthomycin A (Lee et al., 1994). Anderson et al. (1980) demonstrated that [carboxy-13C] AHBA could be efficiently and specifically incorporated into the C-6 methyl group of porfiromycin, which contains the same mitosane core as MC. Incorporation experiments with radiolabeled precursors have demonstrated that the mitosane core of MC was derived from the junction of AHBA and D-glucosamine (Anderson et al., 1980; Hornemann, 1981).
Meanwhile the Oxe2x80x94 and Nxe2x80x94 (but not Cxe2x80x94) methyl groups were shown to be derived from L-methionine, while the C-10 carbamoyl group came from L-arginine or L-citrulline (Bezanson and Vining, 1971; Hornemann and Eggert, 1975; Hornemann et al., 1974). [14C]-labeled precursor feeding studies with D-glucose, pyruvate and D-erythrose indicated that de novo biosynthesis of AHBA resulted directly from the shikimate pathway. However, no incorporation into the mC7N unit of either MC (Hornemann, 1981) or the ansamycin antibiotics (Chiao et al., 1998) was found from labeling studies with shikimic acid, the shikimate precursor 3-dehydroquinic acid, or the shikimate derived amino acids. These results led to the hypothesis of a modified shikimate pathway, in which a 3-deoxy-D-arabino-heptulosonic acid-7-phosphate (DAHP) synthase-like enzyme catalyzes the conversion to 3,4-dideoxy-4-amino-D-arabino-heptulosonic acid-7-phosphate (amino-DAHP), to give the ammoniated shikimate pathway (Kim et al., 1992). Floss (1997) provided strong support for this new variant of the shikimate pathway by showing that aminoDAHP, 5-deoxy-5-amino-3-dehydroquinic acid (aminoDHQ), and 5-deoxy-5-amino-3-dehydroshikimic acid (aminoDHS) could be efficiently converted into AHBA by a cell-free extract of Amycolatopsis mediterranei (rifamycin producer), in contrast to the normal shikimate pathway intermediate DAHP which was not converted (Kim et al., 1992; Kim et al., 1996). Recently, the AHBA synthase (rifK) gene from A. mediterranei has been cloned, sequenced and functionally characterized (Kim et al., 1998).
Little is known regarding the details of the convergent assembly of MC from AHBA and D-glucosamine in S. lavendulae, i.e., whether its de novo biosynthesis is related to the primary metabolic shikimate pathway, an important route in microorganisms and plants for aromatic amino acid biosynthesis (Floss, 1997). In addition, it is unclear how S. lavendulae resists the activity of MC since the preferred MC alkylation sites in DNA are guanine and cytosine, and MC-induced cell death can result from a single crosslink per genome (Tomasz, 1995).
Thus, there is a continuing need for the identification and isolation of antibiotic biosynthetic genes, including genes which confer resistance to antibiotics or result in enhanced production of antibiotics.
The present invention provides an isolated and purified nucleic acid molecule, e.g., DNA, comprising a gene cluster for mitomycin, a variant or a fragment thereof (the mit/mmc gene cluster). As described hereinbelow, the S. lavendulae mitomycin gene cluster includes the mitomycin biosynthetic gene cluster comprising 47 mitomycin biosynthetic genes spanning 55 kb of contiguous DNA. The biosynthetic portion of the gene cluster includes genes that encode polypeptides involved in the generation of biosynthetic precursors, mitosane ring system assembly and functionalization (e.g., methylation, hydroxylation, aminotransfer, carbamoylation, and carbonyl reduction), a mitomycin resistance gene which is different than mrd and the unlinked mcr, as well as several regulatory genes. Gene disruption was employed to further characterize some of the genes. Fourteen of 22 gene disruption mutants affected mitomycin biosynthesis, resulting in abrogation or overexpression of drug production, e.g., targeted genetic disruption of a mitomycin pathway regulator (e.g., mmcW) led to a substantial increase in drug production. It is preferred that the isolated and purified nucleic acid molecule of the invention is nucleic acid from Streptomyces spp., such as Streptomyces lavendulae (e.g., B19/ATCC 27422, NRRL 2564, KY681, ATCC 27423, or PB1000), Streptomyces caespitosus, Streptomyces verticillatus, and Streptomyces sandaensis (FERM-P7654), although isolated and purified nucleic acid molecules from other organisms which produce mitomycin or biological or functional equivalents thereof are also within the scope of the invention. The nucleic acid molecules of the invention are double-stranded or single-stranded.
As described hereinbelow, a 3.8 kb BamHI fragment from the S. lavendulae genome was isolated which comprises three open reading frames (ORFs). One of the ORFs (mitA) showed high similarity to previously identified AHBA synthase genes (Kim et al., 1998), while another (mitB) showed sequence similarity to several prokaryotic and eukaryotic glycosyltransferases. Nucleotide sequence analysis showed that mitA encodes a 388 amino acid protein that has 71% identity (80% similarity) with the rifamycin AHBA synthase from Amycolatopsis mediterranei, as well as with two additional AHBA synthases from related ansamycin antibiotic-producing microorganisms. Gene disruption and site-directed mutagenesis of the S. lavendulae chromosomal copy of mitA completely blocked the production of MC. The function of mitA was confirmed by complementation of a S. lavendulae strain containing a K191A mutation in MitA with 3-amino-5-hydroxybenzoic acid, i.e., MC production was restored when the mitA mutant strain was cultured in the presence of exogenous 3-amino-5-hydroxybenzoic acid. mitB encodes a 272 amino acid protein.
Seven gene products (aminoDHQ synthase (MitP), aminoquinate dehydrogenase (MitT), aminoDHQ dehydratase (MmcF), AHBA synthase (MitA), oxidoreductase (MitG), phosphatase (MitJ), and kinase (MitS)) are likely responsible for assembly of the intermediate 3-amino-5-hydroxybenzoic acid (AHBA) through a variant of the shikimate pathway. However, the gene encoding aminoDAHP synthase, the first presumed enzyme involved in AHBA biosynthesis from phosphoenol pyruvate (PEP) and erythrose 4-phosphate (E4P), is not linked within the mitomycin biosynthetic gene cluster.
A mitomycin resistance determinant (mct) encodes a membrane-associated protein involved in excretion of mitomycin from cells. Disruption of mct by insertional inactivation resulted in a S. lavendulae mutant strain that was considerably more sensitive to MC. Expression of mct in E. coli conferred a 5-fold increase in cellular resistance to MC, led to the synthesis of a membrane associated protein, and correlated with reduced intracellular accumulation of drug. Co-expression of mct and mrd in E. coli resulted in a 150-fold increase in resistance, as well as reduced intracellular accumulation of MC. The results establish that MRD maintains a high affinity for MC and may serve as the primary receptor (participating as an accessory component in a drug export system) for subsequent transport by MCT.
The cloned mitomycin biosynthetic genes are useful to elucidate the molecular basis for the biosynthesis of the mitosane ring system, as well as to. engineer the biosynthesis of novel natural products. Moreover, genetic engineering or overexpression of the transport, resistance and regulatory proteins may lead to higher titers of mitomycin compounds from production cultures.
Preferably, the isolated nucleic acid molecule comprising the gene cluster includes a nucleic acid sequence comprising SEQ ID NO:96 or SEQ ID NO:76, a variant or a fragment thereof, e.g., a nucleic acid molecule that hybridizes under moderate, or more preferably stringent, hybridization conditions to SEQ ID NO:96, SEQ ID NO:76 or a fragment thereof. Moderate and stringent hybridization conditions are well known to the art, see, for example sections 9.47-9.51 of Sambrook et al. (Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. (1989). For example, stringent conditions are those that (1) employ low ionic strength and high temperature for washing, for example, 0.015 M NaCl/0.0015 M sodium citrate (SSC); 0.1% sodium lauryl sulfate (SDS) at 50xc2x0 C., or (2) employ a denaturing agent such as formamide during hybridization, e.g., 50% formamide with 0.1% bovine serum albumin/0.1% Ficoll/0.1% polyvinylpyrrolidone/50 mM sodium phosphate buffer at pH 6.5 with 750 mM NaCl, 75 mM sodium citrate at 42xc2x0 C. Another example is use of 50% formamide, 5xc3x97SSC (0.75 M NaCl, 0.075 M sodium citrate), 50 mM sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5xc3x97Denhardt""s solution, sonicated salmon sperm DNA (50 xcexcg/ml), 0.1% sodium dodecylsulfate (SDS), and 10% dextran sulfate at 42xc2x0 C., with washes at 42xc2x0 C. in 0.2xc3x97SSC and 0.1% SDS.
A preferred nucleic molecule of the invention comprises a nucleic acid sequence encoding a polypeptide including, but not limited to, MitA (e.g., SEQ ID NO:10 encoded by SEQ ID NO:97), MitB (e.g., SEQ ID NO:11 encoded by SEQ ID NO:98), MitC (e.g., SEQ ID NO:12 encoded by SEQ ID NO:99), MitD (e.g., SEQ ID NO:100 encoded by SEQ ID NO:45), MitE (e.g., SEQ ID NO:101 encoded by SEQ ID NO:44), MitF (e.g., SEQ ID NO:102 encoded by SEQ ID NO:43), MitG (e.g., SEQ ID NO:103 encoded by SEQ ID NO:42), MitH (e.g., SEQ ID NO:104 encoded by SEQ ID NO:41), MitI (e.g., SEQ ID NO:105 encoded by SEQ ID NO:40), MitJ (e.g., SEQ ID NO:106 encoded by SEQ ID NO:39), MitK (e.g., SEQ ID NO:107 encoded by SEQ ID NO:38), MitL (e.g., SEQ ID NO:108 encoded by SEQ ID NO:37), MitM (e.g., SEQ ID NO:109 encoded by SEQ ID NO:36), MitN (e.g., SEQ ID NO:110 encoded by SEQ ID NO:35), MitO (e.g., SEQ ID NO:111 encoded by SEQ ID NO:34), MitP (e.g., SEQ ID NO:112 encoded by SEQ ID NO:33), MitQ (e.g., SEQ ID NO:113 encoded by SEQ ID NO:32), MitR (e.g., SEQ ID NO:114 encoded by SEQ ID NO:31), MitS (e.g., SEQ ID NO:115 encoded by SEQ ID NO:30), MitT (e.g., SEQ ID NO:140 encoded by SEQ ID NO:29), MmcA (SEQ ID NO:116 encoded by SEQ ID NO:49), MmcB (SEQ ID NO:117 encoded by SEQ ID NO:50), MmcC (SEQ ID NO:118 encoded by SEQ ID NO:51), MmcD (SEQ ID NO:119 encoded by SEQ ID NO:52), MmcE (SEQ ID NO:120 encoded by SEQ ID NO:53), MmcF (SEQ ID NO:121 encoded by SEQ ID NO:54), MmcG (SEQ ID NO:122 encoded by SEQ ID NO:55), MmcH (SEQ ID NO:123 encoded by SEQ ID NO:56), Mmcl (SEQ ID NO:124 encoded by SEQ ID NO:57), MmcJ (SEQ ID NO:125 encoded by SEQ ID NO:58), MmcK (SEQ ID NO:126 encoded by SEQ ID NO:59), MmcL (SEQ ID NO:127 encoded by SEQ ID NO:60), MmcM (SEQ ID NO:128 encoded by SEQ ID NO:61), MmcN (SEQ ID NO:129 encoded by SEQ ID NO:62), MmcO (SEQ ID NO:130 encoded by SEQ ID NO:63), MmcP (SEQ ID NO:131 encoded by SEQ ID NO:64), MmcQ (SEQ ID NO:132 encoded by SEQ ID NO:65), MmcR (SEQ ID NO:133 encoded by SEQ ID NO:66), MmcS (SEQ ID NO:134 encoded by SEQ ID NO:67), MmcT (SEQ ID NO:135 encoded by SEQ ID NO:68), MmcU (SEQ ID NO:136 encoded by SEQ ID NO:69), MmcV (SEQ ID NO:137 encoded by SEQ ID NO:70), MmcW (SEQ ID NO:138 encoded by SEQ ID NO:71), MmcX (SEQ ID NO:139 encoded by SEQ ID NO:72), MmcY (SEQ ID NO:141 encoded by SEQ ID NO:73), Mct (SEQ ID NO:17 encoded by SEQ ID NO:16), a variant or a fragment thereof, e.g., a nucleic acid molecule that hybridizes under moderate, or more preferably stringent, hybridization conditions to at least one of the nucleic acid sequences identified hereinabove.
The invention further provides an isolated and purified nucleic acid molecule which is linked to a mitomycin biosynthetic gene cluster and which encodes polyketide biosynthetic enzymes, a variant or a fragment thereof. Preferably, the nucleic acid molecule of this embodiment of the invention comprises at least one, preferably at least five, and more preferably at least nine, open reading frames. More preferably, the nucleic acid molecule hybridizes under moderate, or more preferably stringent, hybridization conditions to SEQ ID NO:74, or a portion thereof.
The invention also provides an isolated and purified nucleic acid molecule which is linked to a mitomycin biosynthetic gene cluster and which encodes sugar biosynthetic enzymes, a variant or a fragment thereof. Preferably, the nucleic acid molecule of this embodiment of the invention comprises at least one, preferably at least five, more preferably at least nine, and even more preferably at least twelve, open reading frames. Preferably, the nucleic acid molecule of this embodiment of the invention hybridizes under moderate, or more preferably stringent, hybridization conditions to SEQ ID NO:75, or a portion thereof.
The invention also provides a variant polypeptide having at least about 80%, more preferably at least about 90%, and even more preferably at least about 95%, but less than 100%, contiguous amino acid sequence identity to a polypeptide having an amino acid sequence encoded by SEQ ID NO:76, or a fragment thereof. A preferred variant polypeptide includes a variant polypeptide or fragment thereof having at least about 1%, more preferably at least about 10%, and even more preferably at least about 50%, the activity of the polypeptide having the amino acid sequence comprising SEQ ID NO:10-12, 17 or 100-141. Thus, for example, the activity of a polypeptide having SEQ ID NO:98 can be compared to a variant of SEQ ID NO:98 having at least one amino acid substitution, insertion, or deletion relative to SEQ ID NO:98.
A variant nucleic acid sequence of the invention has at least about 80%, more preferably at least about 90%, and even more preferably at least about 95%, but less than 100%, contiguous nucleic acid sequence identity to a nucleic acid sequence comprising SEQ ID NO:76, or a fragment thereof. The amino acid and/or nucleic acid similarity (or homology) of two sequences may be determined manually or using algorithms well known to the art.
The invention also provides probes and primers comprising at least a portion of the nucleic acid molecules of the invention. The probes or primers of the invention are preferably detectably labeled or have a binding site for a detectable label. Preferably, the probes or primers of the invention are at least about 7, more preferably at least about 15, contiguous nucleotides bases having at least about 80% identity, more preferably at least about 90% identity, to the isolated nucleic acid molecules of the invention. Such probes or primers are useful to detect, quantify, isolate and/or amplify DNA strands with complementary to sequences related to the mitomycin biosynthetic gene cluster, sequences related to those encoding the polyketide biosynthetic enzymes linked to the mitomycin biosynthetic gene cluster, sequences related to those encoding sugar biosynthetic enzymes linked to the mitomycin biosynthetic gene cluster, a variant or a fragment thereof
Also provided is an expression cassette comprising a nucleic acid molecule comprising at least a portion of a mitomycin biosynthetic gene cluster, a nucleic acid molecule which is linked to a mitomycin biosynthetic gene cluster and which encodes polyketide biosynthetic enzymes, a nucleic acid molecule which is linked to a mitomycin biosynthetic gene cluster and which encodes sugar biosynthetic enzymes, a variant or fragment thereof, operably linked to a promoter functional in a host cell. Host cells that have been modified genetically, i.e., recombinant host cells, include host cells comprising an expression cassette, e.g., an expression cassette of the invention, or host cells in which the genome has been genetically manipulated, e.g., by deletion of a portion of, replacement of a portion of, or by disruption of, the host chromosome, so as to reduce or eliminate the expression of a particular mitomycin biosynthetic gene, polyketide biosynthetic gene or a sugar biosynthetic gene of the invention.
One embodiment of the invention is a recombinant host cell, e.g., a bacterial cell, in which a portion of a nucleic acid sequence comprising the mitomycin gene cluster, i.e., the endogenous or native genomic sequence, is disrupted or replaced, for example, by an insertion with heterologous. sequences or substituted with a variant nucleic acid sequence of the invention, preferably so as to result in altered mitomycin synthesis, such as an increase in mitomycin synthesis, and/or production of a novel compound. For example, the invention includes a recombinant host cell in which the mmcW gene is disrupted, for example, by replacement with a selectable marker gene, so as to yield a recombinant host cell having an increase in mitomycin production.
Another embodiment of the invention is a recombinant host cell, the genome of which is augmented by an expression cassette, e.g., via an extrachromosomal element such as a plasmid or by stable integration of the cassette into the host chromosome. Thus, the genome of the recombinant host cell is augmented with at least one mitomycin biosynthetic gene, polyketide biosynthetic gene or a sugar biosynthetic gene of the invention so as to yield an altered level of mitomycin and/or a novel compound(s) relative to the corresponding non-recombinant host cell.
Alternatively, the genome of a recombinant host cell is augmented with a non-mitomycin biosynthetic gene and, optionally, at least one mitomycin biosynthetic gene, polyketide biosynthetic gene or a sugar biosynthetic gene of the invention so as to yield an altered level of mitomycin and/or a novel compound(s) relative to the corresponding non-recombinant host cell. For example, the recombinant host cell may be augmented with pika (see U.S. application Ser. No. 09/105,537, filed Jun. 26, 1998, the disclosure of which is incorporated by reference herein) and pikA expressed in an amount effective to yield a novel compound(s).
Host cells useful to prepare the recombinant host cells of the invention include cells which do not express or do not comprise nucleic acid corresponding to the nucleic acid molecules of the invention, e.g., mitomycin biosynthetic genes, as well as cells which naturally produce mitomycin.
Thus, the invention also provides isolated and purified polypeptides encoded by a nucleic acid molecule of the invention. Preferably, the polypeptide of the invention is obtained from recombinant host cells, e.g., the genome of which is augmented by a nucleic acid molecule of the invention. In addition, expression cassettes and host cells comprising antisense sequences of at least a portion of the mitomycin biosynthetic gene cluster of the invention are envisioned.
In another embodiment of the invention, the isolated and purified nucleic acid molecule which is linked to a mitomycin biosynthetic gene cluster and which encodes polyketide biosynthetic enzymes, e.g., a polyketide synthase, is useful in methods to prepare recombinant polyhydroxyalkanoate monomer synthases and polymers.
Thus, the present invention provides a method of preparing a polyhydroxyalkanoate synthase. The method comprises introducing an expression cassette into a host cell. The expression cassette comprises a DNA molecule encoding a polyketide synthase, operably linked to a promoter functional in the host cell. The DNA molecule is preferably obtained from a mitomycin-producing organism, e.g., a Streptomyces spp. such as S. lavendulae. The DNA molecule encoding the polyketide synthase is then expressed in the cell. Thus, another embodiment of the invention provides a purified recombinant polyketide isolated from a host cell which expresses the synthase.
Another embodiment of the invention is a method of preparing a polyhydroxyalkanoate polymer. The method comprises introducing a first expression cassette and a second expression cassette into a host cell. The first expression cassette comprises a DNA segment encoding a fatty acid synthase in which the dehydrase activity has been inactivated that is operably linked to a promoter functional in the host cell, e.g., an insect cell. The inactivation preferably is via a mutation in the catalytic site of the dehydrase. The second expression cassette comprises a DNA segment encoding a polyketide synthase that is preferably obtained from a mitomycin-producing organism operably linked to a promoter functional in the host cell. The expression cassettes may be on the same or separate molecules. The DNA segments in the expression cassettes are expressed in the cell so as to yield a polyhydroxyalkanoate polymer.
The present invention also. provides an expression cassette comprising a nucleic acid molecule encoding a polyhydroxyalkanoate monomer synthase operably linked to a promoter functional in a host cell. The nucleic acid molecule comprises a plurality of DNA segments. Thus, the nucleic acid molecule comprises at least a first and a second DNA segment. The first DNA segment encodes a first module and the second DNA segment encodes a second module, wherein the DNA segments together encode a polyhydroxyalkanoate monomer synthase. No more than one DNA segment is derived from the eryA gene cluster of Saccharopolyspora erythraea. It is also preferred that the first DNA segment comprises a module from a mitomycin-producing organism, e.g., Streptomyces spp. The nucleic acid molecule may optionally further comprise a third DNA segment encoding a polyhydroxyalkanoate synthase. Alternatively, a second nucleic acid molecule encoding a polyhydroxyalkanoate synthase may be introduced into the host cell.
Also provided is an isolated and purified DNA molecule. The DNA molecule comprises a plurality of DNA segments. Thus, the DNA molecule comprises at least a first and a second DNA segment. The first DNA segment encodes a first module and the second DNA segment encodes a second module. Together the DNA segments encode a recombinant polyhydroxyalkanoate monomer synthase. It is preferred that no more than one DNA segment is derived from the eryA gene cluster of Saccharopolyspora erythraea. Also, it is preferred that no more than one module is derived from the gene cluster from Streptomyces hygroscopicus that encodes rapamycin or the gene cluster that encodes spiramycin. A preferred embodiment of the invention employs a first DNA segment comprising a module from a mitomycin-producing organism. A further preferred embodiment of the isolated DNA molecule of the invention includes a DNA segment encoding a polyhydroxyalkanoate synthase.
Further provided is a method of preparing a polyhydroxyalkanoate polymer. The method comprises introducing a first DNA molecule and a second DNA molecule into a host cell. The first DNA molecule comprises a DNA segment encoding a recombinant polyhydroxyalkanoate monomer synthase. The recombinant polyhydroxyalkanoate monomer synthase comprises a plurality of modules. Thus, the monomer synthase comprises at least a first module and a second module. The first DNA molecule is operably linked to a promoter functional in a host cell. The second DNA molecule comprises a DNA segment encoding a polyhydroxyalkanoate synthase operably linked to a promoter functional in the host cell. It is preferred that at least one module is from a mitomycin-producing organism. The DNAs encoding the recombinant polyhydroxyalkanoate monomer synthase and polyhydroxyalkanoate synthase are expressed in the host cell so as to generate a polyhydroxyalkanoate polymer.
Yet another embodiment of the invention is an isolated and purified DNA molecule. The DNA molecule comprises a plurality of DNA segments. That is, the DNA molecule comprises at least a first and a second DNA segment. The first DNA segment encodes a fatty acid synthase and the second DNA segment encodes a module of a polyketide synthase. A preferred embodiment of the invention employs a second DNA segment comprising a module of a polyketide synthase from a mitomycin-producing organism such as Streptomyces.
Also provided is a method of providing a polyhydroxyalkanoate monomer synthase. The method comprises introducing an expression cassette into a host cell. The expression cassette comprises a DNA molecule encoding a polyhydroxyalkanoate monomer synthase operably linked to a promoter functional in the host cell. The monomer synthase comprises a plurality of modules. Thus, the monomer synthase comprises at least a first and second module which together encode the monomer synthase. A preferred embodiment of the invention employs a module from a mitomycin-producing organism. Optionally, the expression cassette further comprises a second DNA molecule encoding a polyhydroxyalkanoate synthase.
The invention also provides an isolated and purified DNA molecule comprising a first DNA segment encoding a first module and a second DNA segment encoding a second module, wherein the DNA segments together encode a recombinant polyhydroxyalkanoate monomer synthase. Preferably, at least one DNA segment is derived from DNA which is linked to the mitomycin gene cluster of S. lavendulae. Also preferably, no more than one DNA segment is derived from the eryA gene cluster of Saccharopolyspora erythraea. In one embodiment of the invention, the 3xe2x80x2 most DNA segment of the isolated DNA molecule of the invention encodes a thioesterase II. Also provided is an expression cassette comprising a nucleic acid molecule encoding the polyhydroxyalkanoate monomer synthase operably linked to a promoter functional in a host cell.
Yet another embodiment of the invention is a method of providing a polyhydroxyalkanoate monomer. The method comprises introducing into a host cell a DNA molecule comprising a DNA segment encoding a recombinant polyhydroxyalkanoate monomer synthase operably linked to a promoter functional in the host cell. Preferably, the second DNA molecule is derived from DNA which is linked to the mitomycin gene cluster. The recombinant polyhydroxyalkanoate monomer synthase comprises a first module and a second module, wherein at least one DNA segment is derived from DNA which is linked to a mitomycin gene. cluster, e.g., the mitomycin gene cluster of S. lavendulae. The DNA encoding the recombinant polyhydroxyalkanoate monomer synthase is then expressed in the host cell so as to generate a polyhydroxyalkanoate monomer. Optionally, a second DNA molecule may be introduced into the host cell. The second DNA molecule comprises a DNA segment encoding a polyhydroxyalkanoate synthase operably linked to a promoter functional in the host cell. The two DNA molecules are expressed in the host cell so as to generate a polyhydroxyalkanoate polymer.
Another embodiment of the invention is an isolated and purified DNA molecule comprising a first DNA segment encoding a fatty acid synthase and a second DNA segment encoding a module from the DNA which is linked to the mitomycin gene cluster of S. lavendulae. Such a DNA molecule can be employed in a method of providing a polyhydroxyalkanoate monomer. Thus, a DNA molecule comprising a first DNA segment encoding a fatty acid synthase and a second DNA segment encoding a polyketide synthase is introduced into a host cell. The first DNA segment is 5xe2x80x2 to the second DNA segment and the first DNA segment is operably linked to a promoter functional in the host cell. The first DNA segment is linked to the second DNA segment so that the linked DNA segments express a fusion protein. The DNA molecule is expressed in the host cell so as to generate a polyhydroxyalkanoate monomer.
Further provided is a method of providing a polyhydroxyalkanoate monomer synthase. The method comprises introducing an expression cassette comprising a DNA molecule encoding a polyhydroxyalkanoate synthase operably linked to a promoter functional in a host cell. The DNA molecule comprises a first DNA segment encoding a first module and a second DNA segment encoding a second module wherein the DNA segments together encode a polyhydroxyalkanoate monomer synthase. At least one DNA segment is derived from DNA which is linked to the mitomycin gene cluster of S. lavendulae. The DNA molecule is expressed in the host cell. Optionally, the DNA molecule further comprises a DNA segment encoding a polyhydroxyalkanoate synthase. Alternatively, a second, separate DNA molecule encoding a polyhydroxyalkanoate synthase is introduced into the host cell.
Thus, the invention provides an isolated and purified DNA molecule comprising a first DNA segment encoding a first module and a second DNA segment encoding a second module, wherein the DNA segments together encode a recombinant polyhydroxyalkanoate monomer synthase, and wherein at least one DNA segment is derived from the mit/mmc gene cluster of S. lavendulae. Preferably, no more than one DNA segment is derived from the eryA gene cluster of Saccharopolyspora erythraea. In one embodiment of the invention, the 3xe2x80x2 most DNA segment of the isolated DNA molecule of the invention encodes a thioesterase II. Also provided is an expression cassette comprising a nucleic acid molecule encoding the polyhydroxyalkanoate monomer synthase operably linked to a promoter functional in a host cell.
Yet another embodiment of the invention is a method of providing a polyhydroxyalkanoate monomer. The method comprises introducing into a host cell a DNA molecule comprising a DNA segment encoding a recombinant polyhydroxyalkanoate monomer synthase operably linked to a promoter functional in the host cell. The recombinant polyhydroxyalkanoate monomer synthase comprises a first module and a second module, wherein at least one DNA segment is derived from the mit/mmc gene cluster of S. lavendulae. The DNA encoding the recombinant polyhydroxyalkanoate monomer synthase is then expressed in the host cell so as to generate a polyhydroxyalkanoate monomer. Optionally, a a second DNA molecule may be introduced into the host cell. The second DNA molecule comprises a DNA segment encoding a polyhydroxyalkanoate synthase operably linked to a promoter functional in the host cell. The two DNA molecules are expressed in the host cell so as to generate a polyhydroxyalkanoate polymer.
Another embodiment of the invention is an isolated and purified DNA molecule comprising a first DNA segment encoding a fatty acid synthase and a second DNA segment encoding a module from the mit/mmc gene cluster of S. lavendulae. Such a DNA molecule can be employed in a method of providing a polyhydroxyalkanoate monomer. Thus, a DNA molecule comprising a first DNA segment encoding a fatty acid synthase and a second DNA segment encoding a polyketide synthase is introduced into a host cell. The first DNA segment is 5xe2x80x2 to the second DNA segment and the first DNA segment is operably linked to a promoter functional in the host cell. The first DNA segment is linked to the second DNA segment so that the linked DNA segments express a fusion protein. The DNA molecule is expressed in the host cell so as to generate a polyhydroxyalkanoate monomer.
Further provided is a method of providing a polyhydroxyalkanoate monomer synthase. The method comprises introducing an expression cassette comprising a DNA molecule encoding a polyhydroxyalkanoate synthase operably linked to a promoter functional in a host cell. The DNA molecule comprises a first DNA segment encoding a first module and a second DNA segment encoding a second module wherein the DNA segments together encode a polyhydroxyalkanoate monomer synthase. At least one DNA segment is derived from the mit/mmc gene cluster of S. lavendulae. The DNA molecule is expressed in the host cell. Optionally, the DNA molecule further comprises a DNA segment encoding a polyhydroxyalkanoate synthase. Alternatively, a second, separate DNA molecule encoding a polyhydroxyalkanoate synthase is introduced into the host cell.
Also provided is a method for directing the biosynthesis of specific sugar-modified polyketides by genetic manipulation of a polyketide-producing microorganism. The method comprises introducing into a polyketide-producing microorganism a DNA sequence encoding enzymes in sugar biosynthesis, e.g., a DNA sequence comprising SEQ ID NO:75, a variant or fragment thereof, so as to yield a microorganism. that produces specific sugar-modified polyketides. Alternatively, an anti-sense DNA sequence of the invention may be employed. Then the sugar-modified polyketides are isolated from the microorganism. It is preferred that the DNA sequence is modified so as to result in the inactivation of at least one enzymatic activity in sugar biosynthesis or in the attachment of the sugar to a polyketide
Thus, the modules encoded by the nucleic acid segments of the invention may be employed in the methods described hereinabove to prepare polyhydroxyalkanoates of varied chain length or having various side chain substitutions.
The compounds produced by the recombinant host cells of the invention are preferably biologically active agents such as antibiotics, anti-inflammatory agents, anti-cancer agents, antibiotics, immune-enhancers, immunosuppressants, agents to treat asthma, chronic obstructive pulmonary disease as well as other diseases involving respiratory inflammation, or cholesterol-lowering agents; or as crop protection agents (e.g., fungicides or insecticides), as well as biopolymers, e.g., in packaging or biomedical applications, or to engineer PHA monomer synthases. Methods employing these compounds, e.g., to treat a mammal, e.g., a human, bird or fish in need of such therapy, are also envisioned.